VAMP-8 acts at a late fusion step in the mast cell degranulation response. (A) The levels of SNARE and SNARE-related proteins in VAMP-8–deficient and WT BMMCs were analyzed. An equivalent of 2 × 10⁵ BMMCs per lane was migrated on SDS-PAGE and analyzed by Western blotting using indicated specific Abs. (B) VAMP-8–deficient and WT BMMCs were stimulated with either thapsigargin (1 μM) or ionomycin/PMA (500 nM/20 nM) for 10 minutes, and percentage release of β-hexosaminidase was determined. Data are means plus or minus SEM from triplicate samples derived from 3 different BMMC cultures and are representative of 3 independent experiments. **P < .05 by one-way ANOVA. (C) Enhanced association of SNAP-23 and syntaxin-4 with VAMP-8 after stimulation. IgE-sensitized indicated BMMCs (10⁷) were either left unstimulated (NS) or were stimulated (S) with 10 ng/mL DNP-HSA for indicated times followed by NEM treatment as described in “Immunoblotting and immunoprecipitation.” Immunoprecipitation was performed with indicated Abs followed by immunoblotting with anti–VAMP-8. Blots were then stripped and reprobed with Abs to either syntaxin-4 or SNAP-23 as indicated. Corresponding quantitative analysis is shown to the right (n = 3, **P < .05). (D) VAMP-8–deficient BMMCs show a tendency to increased complex formation with SNAP-23 in stimulated BMMCs. IgE-sensitized indicated BMMCs (10⁷) were treated as described in panel C. Immunoprecipitation was performed with SNAP-23 Abs followed by immunoblotting with anti–VAMP-2. For loading control, blots were cut into 2 parts and probed with SNAP-23 as indicated. Corresponding quantitative analysis is shown to the right (n = 3).